Influenza viruses are generally divided into three types: A, B, and C, based on the antigenic differences between their nucleoprotein and matrix protein antigens. Influenza A viruses are further divided into subtypes depending on the antigenic nature of the two major viral surface proteins, the hemagglutinin (HA) and neuraminidase (NA) proteins. Currently, 15 subtypes of HA are known (Lamb and Krug. 2001). Both HA and NA carry antigenic epitopes. Antibodies that are raised against HA and NA are associated with resistance to infection and/or illness in humans and animals. The efficacy of a vaccination against influenza is largely determined by the amount of immunogenic HA in a vaccine (Wright and Webster. 2001).
For several decades the HA content of influenza whole-virus and split vaccines derived from this, has been assayed using Single Radial Immunodiffusion (SRID). In this assay, influenza virions are disrupted by detergent and submitted to immunodiffusion for three days at room temperature in antibody-loaded agarose gels. Upon gel staining, the precipitation zone diameters of antigen-antibody complexes are measured, and the antigen content of virus preparations of a certain subtype is calculated by using a calibration curve obtained with a whole virus reference batch of this subtype (NIBSC, Hertfordshire, UK) with a known HA content (Wood et al. 1977).
However, this SRID assay has a number of disadvantages. Apart from being time consuming, laborious and not leaving room for very high throughput (Wood et al. 1977), the quantification of HA by SRID was shown to be inaccurate when analyzing split vaccines or subunit vaccines (Johannsen et al. 1985). In addition, the virus sample environmental background (its pH and ionic strength) and the choice of detergent for disintegrating the influenza virus and its HA were shown to affect the determination of the HA titer (Willkommen et al. 1983; Bizhanov et al. 1988). Despite all shortcomings of the SRID assay, and calls from experts in the field that in addition to the SRID assay a physico-chemical quantification method should be used for the quantification of HA (Pereira. 1973; Johannsen et al. 1985), immunodiffusion techniques are still the only methods approved by the regulatory authorities for the evaluation of influenza vaccines.
A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method to separate influenza virus components has been described (Phelan and Cohen. 1983). Viral proteins were solubilized and denatured in guanidine-HCl, and reduced by incubation with dithiothreitol (DTT) for several hours at room temperature. It is a well-recognized fact in the art that, under denaturing conditions upon reduction, mature and activated HA0 falls apart in the relatively hydrophilic subunit HA1 and the hydrophobic subunit HA2, the latter still containing the trans membrane domain of the original HA0. Subsequently, analysis was performed by RP-HPLC at room temperature on an (C8) Aquapore column, applying a linear gradient of 0.05% TFA in water to 0.05% TFA in acetonitrile. However, the separation of the various virus components was far from optimal, whereas the recovery was low and not quantitative, presumably due to aggregation of the virus components and/or nonspecific adsorption to the HPLC system/column. In addition, in this HPLC assay HA2 could not be detected, presumably because it had been trapped on the column matrix due to its strong hydrophobic nature.
Kemp et al. (1980) also discloses a method for separating influenza HA using RP-HPLC: radiolabeled tryptic glycopeptides (small parts) of HA are pre-isolated from SDS/PAGE gels and subsequently analyzed by HPLC. The method disclosed by Kemp et al. has the disadvantage of not being suitable for a high-throughput system, because the isolation from gel renders the method rather laborious. Moreover, the chromatographs clearly indicate the poor resolution of the peaks, overlapping with numerous other viral peaks, which makes that the method cannot be used for quantitative purposes. The isolation of numerous bands related to different peptides of different size from gel makes that the method is not suitable for very accurate quantification and repeatability. Moreover, the method of Kemp et al. is not suitable for real-life (non-radiolabeled) samples as the radiolabel is detected, and not suitable for crude sample analyses.
In yet another study (Van der Zee et al. 1983) a method has been disclosed for the purification of Sendai virus envelope proteins using RP-HPLC. Although Van der Zee et al. state that some proteins could be recovered in pure form, this was only assessed by SDS/PAGE, which method is not a very accurate means to show purity of a sample. The chromatograms show that resolution is poor: this indicates that any accurate quantification, based on the HPLC chromatograms is not possible using the purification method disclosed. Moreover, it seems that the detergent interferes with the peak of interest. Furthermore, carry-over of proteins from one analysis to the other is significant. In general, it is clear that the art does not disclose methods and means for an accurate determination of HA concentration in either crude or purified HA samples.
Clearly, there is a strong need for a robust, accurate and fast method for reliable separation and quantification of HA in upstream- and downstream-process preparations, as well as for final vaccine formulations.